Cryptococcus neoformans is a neurotropic pathogen that,unless treated,causes fatal meningoencephalitis primarily in individuals with T-cell deficiency such as the AIDS patients. However, the fungus also causes infection in otherwise normal patients at a low frequency. C. neoformans is an obligate aerobe which is commonly found in the human environment world-wide. The fungus requires atmospheric concentration of oxygen for optimum growth in vitro and the growth is drastically retarded at oxygen levels lower than that of the atmosphere. In the past two years we constructed an insertional mutant library composed of 30,000 mutant clones and screened them for their ability to grow in a hypoxic chamber with 1% oxygen and 5% carbon dioxide. We identified numerous clones that were unable to grow under hypoxic conditions. The mutants belonged to two categories, one had mutations in genes that function in the pathway of SREBP (sterol regulatory element binding protein) and the other category belonged to cyptcococcus specific-gene mutations which have not been known to be associated with the oxygen sensing system. The genes identified in these two categories were deleted in the serotype D reference strain, B-3501. Deletants expressing the expected phenotype (unable to grow under low oxygen) were complemented with their respective wild type genes. These strains are being evaluated for their ability to cause fulminating meningoencephalitis. Simultaneously, all 30000 clones were screened for their ability to grow in the presence of cobalt chloride which has been used as a hypoxia mimicking agent in mammalian systems. We have identified more than 10 mutants that are sensitive to cobalt chloride and all of them exhibit a growth defect under low oxygen conditions at 37 degree celsius.A majority of the mutants were found to be compromised in their mitochondrial function, which is reflected by their reduced rate of respiration. Some of the mutants are also defective in mitochondrial membrane permeability, suggesting the importance of an intact respiratory system for survival under both high concentrations of cobalt chloride as well as low oxygen conditions. In addition, the mutants tend to accumulate intracellular reactive oxygen species and all mutants show sensitivity to various ROS generating chemicals. Gene expression analysis revealed the involvement of several pathways in response to cobalt chloride. Our findings indicate the cobalt chloride sensitivity and/or sensitivity to low oxygen conditions is linked to mitochondrial function, sterol and iron homeostasis, ubiquitination and the ability of cells to respond to ROS. These findings imply that multiple pathways are involved in oxygen sensing in C. neoformans.[unreadable] During the current period, we also screened more than 100 strains of C. neoformans isolated either from environment or clinical specimen before 1975 for thier resistance to fluconazole, a triazole most widely used for the treatment of cryptococcosis as well as for the maintenance therapy of HIV infected patients with cryptococcosis. This approach was taken to determine if the heteroresistance of C. neoformans to fluconazole is intrinsic or related to drug exposure. Since azole drugs were not available until the mid 1980s, these strains are most appropriate for such purpose. Surprisingly, close to 25% of the isolates manifested heteroresistance at 32ug or higher concentrations of fluconazole indicating that the azole heteroresistance in C. neoformans is one of the intrinsic stress management mechanisms.